Detection of gastrin mRNA in human antral mucosa and digestive endocrine tumors by in situ hybridization: a correlative study with immunocytochemistry and electron microscopy.

In gastrinomas, as well as in other endocrine tumors whose hormone overproduction is responsible for clinical syndromes, antibodies against the bioactive form(s) of hormones can fail to detect immunoreactivity. Moreover, tumor secretory granule morphology may fail to allow tumor type identification. The use of anti-pre-pro-gastrin antibodies has been proposed as an alternative to identify gastrinomas. The aim of the present study was to demonstrate that in situ detection of gastrin mRNA may represent another possibility. A 35S-labeled cDNA probe encoding the human gastrin pre-pro-hormone was used to localize gastrin gene transcripts in antral mucosa and digestive endocrine tumors from patients with a Zollinger-Ellison syndrome characterized by high serum gastrin levels. In situ hybridization was combined with light and electron microscopic immunostaining of the bioactive gastrin 17/34 form and morphological study of secretory granules. Gastrin mRNAs were detected in antral gastrin cells and in a variable proportion of tumor cells in all endocrine tumor studied. Transcript expression correlated well with immunohistochemical staining and granule ultrastructure for most of the tumors, and provided crucial evidence for identifying as gastrinomas two tumors with weak immunoreactivity and poorly granulated cells. Our data show that in situ hybridization is a sensitive method for gastrin mRNA detection and represents a valuable tool for the identification of gastrinomas.

Detection and localization by in situ molecular biology techniques and immunohistochemistry of hepatitis C virus in livers of chronically infected patients.

Hepatitis C virus (HCV) detection in the livers of chronically infected patients remains a debatable issue. We used immunohistochemistry, in situ hybridization (ISH) alone or after microwave heating with FITC-labeled probes, RT-PCR with unlabeled primers followed by ISH (RT-PCR-ISH), and in situ RT-PCR with FITC-labeled primers (in situ RT-PCRd) to localize the virus in 38 liver biopsy specimens from 21 chronically infected HCV patients treated with interferon-alpha (IFN-alpha). Biopsies were taken at the beginning and end of IFN-alpha treatment and 1 year later. Results were compared with that of HCV-PCR in serum. RT-PCR-ISH and in situ RT-PCRd showed HCV signal in all liver biopsies even in responders with seronegative HCV PCR. This signal was intranuclear, diffuse, or peripheral, in hepatocytes, bile ductule cells, and lymphocytes. Cytoplasmic signals were occasionally observed. Whereas the percentage of labeled hepatocytes remained constant, the number of labeled lymphoid follicles decreased after INF-alpha therapy. Immunohistochemistry resulted in the same pattern of positivity but it was weaker and inconstant. This study indicates the persistency of HCV latency in IFN-alpha responders 1 year after IFN-alpha treatment cessation, a finding that certainly deserves confirmation.

The metabolism of prostaglandins by the guinea-pig uterus with particular reference to corpus luteal maintenance in early pregnancy.

1 Homogenized lung tissue from day 15, pregnant and non-pregnant guinea-pig metabolized exogenous prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) almost completely when incubated in Tyrode solution containing beta-nicotinamide adenine dinucleotide (NAD+). 2 Metabolism of PGE2 and PGF2alpha by homogenates of day 15, pregnant or non-pregnant, guinea-pig uteri was relatively low (8 to 20%) when incubated in Tyrode solution containing NAD+. 3 Day 15, guinea-pig conceptuses (placentae and embryos), homogenized and incubated in Tyrode solution containing NAD+, metabolized prostaglandins to a slightly greater extent (14 to 22%) than day 15, uterine tissue. 4 Addition of exogenous arachidonic acid to day 12 or day 15, pregnant, guinea-pig uterine homogenates in Tyrode solution did not increase the yield of PGF2alpha following incubation. 5 It is concluded that neither an increase in metabolism of PGF2alpha by the uterus and conceptus nor lack of precursor is responsible for the lower secretion of PGF2alpha from the day 15, pregnant, guinea-pig uterus when compared to the day 15, non-pregnant uterus.

The effect of cloprostenol on human luteal steroid and prostaglandin secretion in vitro.

1 Human luteal tissue slices from days 18, 21 and 25 of the menstrual cycle were superfused in vitro with Medium 199 alone or containing cloprostenol (1 microgram/ml). Concentrations of progesterone, oestradiol-17beta and prostaglandins F2alpha and E2 were determined in the superfusate samples. 2 Secretion of steroids and prostaglandins was maintained at an approximately constant level throughout the experiments (21 h in one case) when the tissue was perfused with M199 alone. 3 Superfusion with cloprostenol (1 microgram/ml) resulted in an initial depression of progesterone and oestradiol-17beta but this was not maintained, levels returning to control values or showing an increase, while superfusion with cloprostenol continued. Cloprostenol is not therefore considered to be luteolytic at this dose and under these conditions for human luteal tissue in vitro. 4 Superfusion with cloprostenol (1 microgram/ml) also resulted in a large stimulation of secretion of endogenous prostaglandin F2 alpha following a short lag phase. This stimulation was possibly due to the initial depression of progesterone secretion. A short-lived stimulation of prostaglandin E2 secretion was also observed. 5 The significance of the increase in prostaglandin E2 secretion and the interrelationships between the various changes observed with cloprostenol are difficult to interpret.

Progesterone, prostaglandin F2alpha, and oestradiol in the utero-ovarian venous plasma of non-pregnant and early, unilaterally pregnant guinea-pigs.

Levels of progesterone, prostaglandin F2alpha (PGF2alpha) and oestradiol in the utero-ovarian venous plasma of non-pregnant and early, unilaterally pregnant guinea-pigs were compared. Progesterone levels fell from day 12 to day 15 in the non-pregnant animals, while levels of PGF2alpha and oestradiol increased during this period. In contrast, in the pregnant guinea-pigs, progesterone levels remained high up to day 15 and PGF2alpha and oestradiol levels remained low in plasma from both the pregnant and non-pregnant sides. These findings support the hypothesis that the guinea-pig conceptus secretes an anti-luteolytic factor in early pregnancy, which reduces the amount of PGF2alpha produced by the uterus and released into the uterine veins and thereby allows luteal maintenance and continued progesterone secretion. Oestradiol, in the presence of progesterone, is regarded as the physiological stimulus for normal uterine PGF2alpha synthesis and release. Consequently, the inhibition of ovarian oestradiol secretion seen in early pregnancy is probably at least part of the mechanism by which the anti-luteolytic factor produced by the conceptus reduces PGF2alpha synthesis by and release from the uterus.

Lactation and fertility in goats after the induction of parturition with an analogue of prostaglandin F2 alpha, cloprostenol.

Cloprostenol, 100 micrograms, given intramuscularly to the nanny, with 50 micrograms 10 hours later, precipitated parturition in goats after 36 +/- 1 hours (mean +/- SEM), when administered at 137 +/- 0.5 days gestation. All kids were born alive and survived to weaning. Milk yield over 40 weeks post partum was not significantly different from that after spontaneous parturition. Three hundred micrograms cloprostenol (200 micrograms with 100 micrograms 10 hours later) also initiated parturition at 137 +/- 0.5 days gestation but caused a significant (P less than 0.01) suppression of lactation. Cloprostenol-induced parturition in more than one pregnancy had no adverse effects except for an increased incidence of placental retention, which was treated successfully with intrauterine pessaries containing oestrogen. During the first eight days after spontaneous parturition efficiency of milk secretion was inversely related to udder mass, suggesting a gradual maturation of the secretory alveolar epithelium over this time. When parturition was induced by cloprostenol there was a four to eight day delay before the establishment of this relationship which appeared essential for a successful lactation. Cloprostenol proved to be a useful tool for the control of parturition in goats, having applications to both general animal husbandry and for the study of mammary development and secretory competence.

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation.

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium-L. japonicus symbiosis.

Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis.

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.

Leader of the pack: faecal pellet deposition order impacts PCR amplification in wombats.

DNA sourced from faeces is notoriously less reliable than that from tissue. Hence, understanding whether faecal pellet quality varies within faecal piles may be important for sample selection. We hypothesized that the order in which faecal pellets are deposited may influence microsatellite polymerase chain reaction (PCR) amplification success from sampled faeces, more specifically, that first pellets deposited will have signatures of greater success than later ones. In a first test of the hypothesis, first and later-deposited pellets, as determined from the direction of footprints, were collected from fresh (overnight) faecal piles of northern hairy-nosed wombats (Lasiorhinus krefftii). DNA extracts were typed for seven microsatellite loci. We found that faecal deposition order significantly affected optical density of bands on autoradiographs (a measure of PCR amplification success) when the first faecal pellet was compared with the last one, but not when the first pellet was only distinguishable from later ones. The absence of a difference in amplification rate between first and later pellets is likely a reflection of the overall high amplification success in this study. That first pellets deposited yield more product suggests they contain more intestinal cells. Although further comparisons are needed, these results may inform sample selection in species for which success of microsatellite PCR amplification of faecal DNA is low. Deposition order may have more of an impact on amplification success and genotyping errors as faecal age increases.

Dimensions of the cranium and of the cranial cavity andintracranial volume in goats (Capra hircus LINNAEUS, 1758)

The aim of this project is to determine the dimensions of the cranium and the cranial cavity and the intracranialvolume in goats, using 64 adults. The dimensions of the cranium and cranial cavity were measured throughmetric tape and paquimeter, considering the intervals of the largest distances. To determine the intracranialvolume, balloons of latex were introduced in the cranial cavity, through the magnum foramen, later on, filledwith water that was transferred for graduate test tube. The average and the standard deviation of length, widthand height of the cranium, in millimeters, were respectively: 218.01 ± 6.96, 120.17 ± 10.01 and 108.14 ± 4.46.The average and the standard deviation of length, width, height, in millimeters, and of the volume of thecranial cavity, in cubic centimeters, were respectively: 109.31 ± 7.25, 61.36 ± 4.51, 63.85 ± 2.88 and119.31 ± 12.21. It was observed that, the width of the cranium possesses positive significant correlationswith the length (r = 0.6865), with the height (r = 0.5644) and with the intracranial volume (r = 0.5436).They were still established, positive significant correlations among the height of the cranial cavity, with thelength (r = 0.5682) and with the intracranial volume (r = 0.5473). Differences were evidenced between malesand females, in relation to the dimensions of the cranium and cranial cavity. There wasn’t difference of theintracranial volume in function of the sex of the goats.

ACTH initiation of mammary secretion in pregnant goats is influenced by the stage of gestation and pre partum milking.

Pregnant goats were treated with ACTH by intramuscular injection (1 mg/d for 2 d) at various stages of pregnancy to investigate its effect on initiation of mammary secretion. ACTH stimulated lactose secretion and increased udder volume at or after 84 d of gestation, when compared with untreated controls. Treatment with ACTH between d 109 and 127 of gestation increased circulating plasma glucose concentration, mammary blood flow and mammary uptake of glucose which returned to pre injection levels 6 d after treatment. Arterial concentrations of progesterone were not affected by ACTH but mammary progesterone uptake increased 2-fold. Pre partum milking alone stimulated mammary secretion in eight of 13 goats. Subsequent treatment with ACTH resulted in no further change in the composition of the secretion but increase in yield was maintained. ACTH stimulated mammary secretion in those goats which did not respond to pre partum milking. It was concluded that ACTH initiated mammary secretion in pregnant goats by mechanisms which interact with the stimulation of milking but which are independent of circulating progesterone levels.

Effect of prostaglandin F-2alpha and uterine extracts on progesterone secretion in vitro by superfused pig corpora lutea.

Corpora lutea obtained from sows in the mid-luteal phase of the oestrous cycle were superfused with various doses of PGF-2alpha, uterine flushings or superfusates from endometrial strips. High doses of PGF-2alpha (1-5 microgram/ml) gave transient stimulation of progesterone secretion; lower doses (0.1-1.0 microgram/ml) inhibited secretion of the steroid, but to a maximum of 55% only and the secretion returned to control levels when superfusion with PGF-2alpha stopped. Uterine flushings from sows in the late luteal phase of the cycle were more effective than mid-cycle flushings in inhibiting progesterone secretion by the luteal tissue: maximal inhibition was 65% and progesterone secretion slowly returned to control levels when superfusion with the flushings was stopped. Superfusate from strips of uterine endometrium from sows in the late luteal phase of the cycle proved most effective in inhibiting progesterone secretion (greater than 90%) and gave a long-lasting inhibition which did not appear to be due to stimulation of PGF-2alpha secretion by the endometrium or corpus luteum.

Antiluteolytic effect of the embryo.

In several mammalian species, prostaglandin F2 alpha (PGF2 alpha) released from the uterus causes corpus luteum regression, and thereby regulates the length of the oestrous cycle or pseudopregnancy. If the animal becomes pregnant, luteal regression must be prevented since ovarian progesterone is necessary for pregnancy maintenance during at least the first one-third of pregnancy. Evidence is presented that the guinea-pig conceptus produces an antiluteolytic factor which suppresses PGF2 alpha output from the uterus by preventing the increase in uterine PGF2 alpha-synthesizing capacity which occurs around the time of luteolysis. Experiments in unilaterally pregnant guinea-pigs, with either an intact or a transected sterile horn, indicate that this factor acts both locally systemically, though separation of the two uterine horns does greatly reduce its effect on the sterile side. Oestradiol administered during early pregnancy induces abortion, which is associated with a high PGF2 alpha-synthesizing capacity of the uterus. However, luteal regression does not take place while the conceptuses are in the uterus. Spontaneously aborting guinea-pigs behave similarly. The conceptus may therefore secrete a luteotrophic hormone as well. Luteal maintenance during early pregnancy in other species is discussed, since experiments on sheep indicate that the sheep embryo may also secrete an antiluteolytic factor.

Local production of prostaglandins in relation to mammary function at the onset of lactation in the goat.

1. Arterial and mammary venous concentrations of prostaglandins F alpha (PGF alpha), E (PGE) and the PGF alpha metabolite, 13,14-dihydro-15-oxoPGF alpha (DHK-PGF alpha) were studied during late pregnancy and the onset of lactation in conscious goats. Mammary secretion concentrations of PGF alpha and DHK-PGF alpha were determined, and mammary blood flow, arterial plasma progesterone concentrations and milk composition were also studied. 2. A significant output of PGF alpha from the mammary gland into mammary venous blood was observed during late pregnancy; this output ceased near term. 3. Mammary output of DHK-PGF alpha into venous blood began about 6 days prepartum, suggesting an increasing capacity of the gland to metabolize PGF alpha. 4. The concentration of PGF alpha in mammary secretion increased from about 4 days pre-partum, that of DKH-PGF alpha from about 12 days pre-partum. 5. It is concluded that although total mammary output of PGF alpha decreases during late pregnancy and early lactation, the rate of mammary synthesis of PGF alpha increases and the PGF alpha is increasingly secreted into milk and metabolized to DHK-PGF alpha within the mammary gland. 6. Unilateral treatment of one mammary gland in goats with the PGF 2 alpha analogue, Cloprostenol, at two dose levels from 2-3 days pre-partum to 1-2 days post-partum prevented the changes in milk [Na] that occur at term in untreated glands. At the higher dose, the normal rise in milk [citrate] was abolished and milk yield was reduced; these effects persisted after cessation of treatment. 7. It is suggested that PGF alpha may play a local inhibitory role in mammary gland function during late pregnancy. It is further suggested that PGF alpha could be the factor, or one of the factors, proposed by Linzell & Peaker (1974) to be responsible for local control of mammary epithelial permeability and possibly also for secretory rate.

Progesterone secretion by the corpus luteum of the early pregnant pig during superfusion in vitro with PGF-2alpha, LH and oestradiol.

Corpora lutea obtained from pregnant sows (Days 18-22) were superfused in vitro for 2-4 h with PGF-2alpha and for 3 h with LH or oestradiol or combinations of PGF-2alpha with LH or oestradiol. LH but not oestradiol stimulated progesterone secretion while superfusion for a minimum of 3 h with PGF-2alpha caused an irreversible decrease in progesterone secretion. LH and PGF-2alpha were mutually antagonistic while oestradiol did not inhibit the luteolytic action of PGF-2alpha.

The metabolism of prostaglandins F2alpha and E2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue, luteal tissue and conceptuses in vitro.

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF2alpha and PGE2 and the degree of metabolism of the prostaglandins measured. Prostaglandins and a metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.

Changes in mammary function at the onset of lactation in the goat: correlation with hormonal changes.

1. Changes in mammary function and plasma hormone concentrations during late pregnancy and the onset of lactation have been studied in conscious goats.2. Mammary blood flow, oxygen consumption and glucose uptake increased markedly and significantly between 2 days and 0.5-1 day pre-partum.3. The increase in mammary glucose uptake was relatively greater than that of oxygen consumption or blood flow.4. The concentration of citrate in mammary secretion increased; the first significant change was apparent 0.5-1 day pre-partum but the main rise occurred after this time.5. It is concluded that the marked increase in mammary glucose uptake 0.5-1 day before parturition indicates the time of onset of copious milk secretion.6. The first significant increase in the concentration of unconjugated oestrogens in arterial plasma occurred 3 days pre-partum, whereas the first significant decrease in progesterone and increase in 13,14-dihydro-15-oxoprostaglandin F(2alpha) occurred 0.5-1 day pre-partum, thus coinciding with the mammary changes; there was also a peak in prolactin concentrations at the latter time.7. The hormonal changes are discussed in relation to current concepts of the initiation of parturition and the onset of copious milk secretion in this species. It is suggested that the fall in plasma progesterone concentrations triggers milk secretion at high rates.